Validation of Real-Time RT-PCR for Detection of SARS-CoV-2 in the Early Stages of the COVID-19 Outbreak Without Emergency Use Authorization Reagents in the Republic of Korea

Background: A real-time reverse transcription polymerase chain reaction (RT-PCR) assay for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed to rapidly diagnose coronavirus disease 2019 (COVID-19). Early diagnosis of COVID-19 enables timely treatment and the implementation of public health measures. We validated the sensitivity, specicity, precision, linearity, accuracy, and robustness of a real-time RT-PCR-based assay for SARS-CoV-2 detection and compared the sensitivity and specicity of Emergency Use Authorization (EUA)-approved reagents for COVID-19 with those of the real-time RT-PCR method. Methods: The real-time RT-PCR-based assay was performed to specically amplify genomic markers of SARS-CoV-2. Results: The real-time RT-PCR assay was highly specic for SARS-CoV-2, and did not amplify genomic fragments of 13 other viruses that cause respiratory diseases. The assay showed high linearity when conducted with a viral isolate from a patient with COVID-19 together with plasmids containing the target genes. The assay showed good repeatability and reproducibility, with a coecient of variation of 3%, and detected SARS-CoV-2 with a limit of detection of 1 PFU/mL. Conclusions: The present real-time RT-PCR-based assay can diagnose COVID-19 with high accuracy and sensitivity. This approach is highly effective and can facilitate the early diagnosis of COVID-19 without the use of EUA reagents in the Republic of Korea.

probe sequences, mixture composition, ampli cation cycles and conditions, and sensitivity. Thus, a standardized, validated assay with highly accurate laboratory performance for detecting SARS-CoV-2 is essential. In this study, the analytical sensitivity, speci city, precision, linearity, accuracy, and robustness of a real-time RT-PCR-based assay for SARS-CoV-2 detection were assessed.
We also compared the sensitivity and speci city of COVID-19 emergency use authorization (EUA)-approved reagents with those of the real-time RT-PCR method investigated in this study. To determine the accuracy of the results obtained using each reagent, evaluation and veri cation were carried out using ve EUA approved products and respiratory samples from suspected patients.

Cells and viruses
The SARS-CoV-2/Korea/KCDC03/2020 virus, isolated by the Korean Center for Disease Control, was used in the experiment. Vero E6 cells were rst inoculated with the viruses and cultured for four days. The culture medium was then centrifuged, aliquoted, and stored at − 70 °C, and the virus titers were measured using a plaque assay. Viral culture was performed in a biosafety level (BSL)-3 laboratory.
Extraction of viral RNA RNA was extracted from the culture medium using a QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Viral lysis was performed in a BSL-3 laboratory, whereas procedures involving RNA were performed in a BSL-2 laboratory. We extracted RNA from 140 µL of the sample using Qiagen viral RNA mini kits, in accordance with the manufacturer's instructions.
Real-time RT-PCR for SARS-CoV-2 Information regarding the primers provided by the WHO and used in real-time RT-PCR [2] is shown in Table 1. AgPath-ID™ one-step RT-PCR reagents (Applied Biosystems, Foster City, CA, USA) were used in accordance with the manufacturer's instructions. One microliter of the primers (10 pmol) and 0.5 µL of probes (10 pmol) were added to the reagents. Primer/probe sets for detecting the RdRp and E genes were added to different tubes. The RNA sample (5 µL) was mixed with the PCR mixture, and PCR was performed at 50 °C for 30 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for one min, for 40 cycles; ROX (carboxyrhodamine) was used as a passive reference dye. The Applied Biosystems™ 7500 Fast Real-Time PCR System was used for real-time RT-PCR with the extracted RNA, and the cycle threshold (Ct) value of the SARS-CoV-2 target gene was ascertained (Table 1).  (Table 2)-and ve samples showing negative results for a known respiratory virus were used. Sensitivity was measured by real-time RT-PCR using plasmids containing the target genes, which were diluted 10-fold from different initial concentrations. To examine the responsivity of the assay to RNA, RT-PCR was performed using 10-fold diluted RNA extracted from a lower respiratory tract sample of the rst patient who tested positive for COVID-19 in South Korea. The cells were incubated at 37 °C for three days to facilitate infection. Two days after viral infection, a 0.03% (w/v) crystal violet overlay was added to each well to stain viable cells.
Determination of intra-assay and inter-assay reproducibility and e ciency To determine the limit of detection (LOD), the titers of the isolated viruses were measured in plaque-forming units (PFU).
The virus culture medium was diluted from 3.45 × 10 6 PFU/mL to 1 × 10 5 PFU/mL. Ten-fold dilutions of the medium were prepared until a concentration of 1 × 10 − 2 PFU/mL was obtained. RNA was extracted from each diluent and used for realtime RT-PCR. The RdRp and E genes were targeted for detection. Real-time RT-PCR was performed in triplicate to assess the assay reproducibility. The assay was repeated three days later, using RNA extracted from the diluted virus culture media to assess repeatability.

Determination of accuracy of EUA reagents
To investigate the accuracy of the COVID-19 EUA-approved reagents in the Republic of Korea (Table 2), 55 positive samples (selected ve-step positive samples based on the distribution of Ct values of the RdRp gene) and 50 negative samples were used to con rm the matching rate (sensitivity and speci city) of the results between the methods used in this study and each EUA product.

Statistical analysis
Inter-assay and intra-assay variations in the Ct value were determined for the triplicate real-time RT-PCR reactions and for the repeat assay three days later. The reliability of each experiment was determined from the F and P values.

Results
Speci city and sensitivity Viral RNA detected by PCR or RT-PCR, which was speci c to the viral genes, was used for the assay. Human coronaviruses 229E, NL63, OC43, and HKU1, SARS-CoV, MERS-CoV, in uenza virus, adenovirus, rhinovirus, parain uenza virus, respiratory syncytial virus, metapneumovirus, and bocavirus were not detected by this assay. The Ct value could not be determined for measles virus, mumps virus, rubella virus, enterovirus D68, or the nasopharyngeal swab specimens. These results indicate that the assay was highly speci c for both target regions, the RdRp and E genes, in SARS-CoV-2 (Table 3). Analytical sensitivity was assessed by determining the LOD for each gene using plasmid DNA containing the RdRp and E genes of SARS-CoV-2. The assay had a mean LOD of 8 × 10 0 copies/µL in triplicate runs ( Evaluation of assay e cacy using a sample from the rst patient with con rmed COVID-19 in South Korea RNA extracted from a lower respiratory tract mucus sample from the rst patient con rmed to have COVID-19 in South Korea was used to evaluate the responsivity of the real-time RT-PCR-based assay for the viral RNA, and to assess its e cacy [4]. Real-time RT-PCR was performed after 10-fold dilution of the RNA. The Ct value of the RdRp gene was 36.62 at a 10 4 -fold dilution, and that of the E gene was 36.97 at 10 − 5 -fold dilution. Real-time RT-PCR accurately detected the target genes in the patient samples (Table 5, Fig. 2).  Fig. 3). * Non-consistent cases in one reagent were con rmed by further examination as inconclusive cases, not false cases ¶ 95% con dence interval: 90.4 ~ 99.7% # 95% con dence interval: 92.9 ~ 100% Limit of detection and limit of quanti cation (correlation between real-time RT-PCR and virus titration) To determine the LOD for SARS-CoV-2, plasmid DNA of a known concentration containing each target gene was used. Assay performance was assessed using 10-fold dilutions of the plasmid DNA standards. Three independent runs were carried out using the plasmid DNA standards and each clone of SARS-CoV-2. The analytical detection limit was 8 × 10 0 copies/µL for all real-time RT-PCR assays (Table 4, Fig. 1). The viral culture medium, with a known viral titer, was diluted from 10 5 to 10 − 2 PFU/mL, and the RNA was extracted from the medium. Real-time RT-PCR was repeated four times to determine the limit of quanti cation at each concentration. The LOD was 1 PFU/mL (Table 5, Fig. 3).

Accuracy and precision
To assess the accuracy and precision of the detection of the target genes using the assay, real-time RT-PCR was performed using four 10-fold serial dilutions of a virus culture medium with a known virus titer. The experiment was repeated three days later. The inter-assay CV was 1.88-2.71 and 0.71-3.47, whereas the intra-assay CV was 0.26-1.21 and 0.16-1.02 for the RdRp and E genes, respectively. The P value was greater than 0.05, and the F value was smaller than 5.99 for all the experiments, indicating accurate and precise detection ( Table 5, Fig. 3).

Accuracy of EUA reagents
Based on the nucleic acid detection reagents evaluated in this study, all ve EUA products could detect SARS-CoV-2 at a sensitivity of at least 98.2% and a speci city of 100% (95% con dence interval: 90.4-99.7%). Non-consistent results obtained with one reagent were con rmed by further examination as inconclusive cases rather than false cases (Table 6).

Discussion
Molecular methods are more rapid, accurate, and sensitive for virus detection than culture methods. In this study, we established a consensus method using molecular tools for detecting SARS-CoV-2. Early diagnosis of SARS-CoV-2-infected patients is essential for controlling the dynamics of the COVID-19 pandemic.
Since its initial emergence in Wuhan, China in late 2019, COVID-19 has rapidly spread worldwide [2]. COVID-19 is caused by SARS-CoV-2, with its clinical symptoms including dyspnea, cough, and mild respiratory symptoms that progress to pneumonia. It is di cult to distinguish SARS-CoV-2 from other common respiratory viruses such as in uenza viruses, because of their highly similar symptoms [8]. A genetic assay with high speci city is necessary to detect SARS-CoV-2.
During early viral spread, the WHO reported a protocol for detecting SARS-CoV-2. The assay was developed by the Charité Research Organization in Germany on January 17, 2020 [9,15]. This was the rst genetic assay to be developed and released after the rst report of the SARS-CoV-2 genome on January 11, 2020. We used this assay to rapidly detect SARS-CoV-2. The protocol involves a common reporter dye, 6-carboxy uorescein FAM, together with BlackBerry Quencher; however, as this quencher is not used in Korea, it was replaced with black hole quencher (BHQ) during probe synthesis. We detected the target genes RdRp and E.
To assess the speci city of the assay, which was performed using speci cally designed primer probes, real-time RT-PCR was conducted on 23 respiratory viruses, including in uenza viruses, and ve respiratory tract samples that had tested negative for SARS-CoV-2. No respiratory viruses other than SARS-CoV-2 were detected. The real-time RT-PCR assay showed excellent sensitivity, and had a high LOD.
Studies have shown that it is possible to accurately detect COVID-19 genes using detection reagents that did not receive EUA. The Provincial Institute of Health and Environmental Research carried out regional training programs for COVID-19 diagnosis, which were particularly useful in emergency diagnostic situations in the early stages of the pandemic. COVID-19 diagnostic EUA reagents were approved for private sector use after comparative analysis.

Conclusions
In conclusion, we evaluated a real-time RT-PCR assay, and found that it had high speci city and sensitivity for SARS-CoV-2 and good analytical performance, using gene cloning and viruses isolated from the rst patient who tested positive for COVID-19 in South Korea. This study was approved by the Korea Centers for Diseases Control and Prevention Ethics Committee-KCDC Authority (approval number #2020-03-01-P-A). This study was performed in accordance with the relevant laws and regulations that Analysis of linearity Real-time PCR for SARS-CoV 2 with RNA from 1st patient's nasopharyngeal swab.

Figure 3
Analysis of Linearity of Real-time PCR for SARS-CoV 2 with RNA form virus isolates.